1. Field of the Invention
The present invention is directed to chemical compounds and methods useful in the regulation of thrombin receptor mediated cell stimulation. More specifically, the invention is directed to prothrombin-derived peptides and methods which employ such peptides for promoting wound healing and inhibiting scar formation, tissue adhesions, blood coagulation, tumor angiogenesis, tumor metastasis and pulmonary edema.
2. Description of the Related Art
Human alpha-thrombin appears to have growth-promoting activity for a wide variety of cells from various tissues. For example, alpha-thrombin has been shown to initiate proliferation of fibroblastic cells in culture without addition of serum or other purified growth factors, to synergize with epidermal growth factor in certain hamster fibroblasts and human endothelial cells, and to initiate cell division or DNA synthesis in mammalian lens epithelial and spleen cells. Yet, the use of thrombin as a growth factor and its potential importance to wound healing has not been widely acclaimed. In part, this may be due to the complexity of thrombin""s involvement with coagulation, platelet activation, and initiation of cell proliferation as well as to the complex regulation of thrombin and thrombin-like molecules by serum protease inhibitors and by cell-released protease nexins. This complexity and high degree of physiologic regulation, however, supports the potential importance of this initiation pathway in wound healing.
Thrombin may also play a role in metastasis and angiogenesis of tumors. Generally, for a tumor to grow larger than a few millimeters in diameter, vascular endothelium must proliferate and form vesicle walls to provide circulation and nutrients to the cells inside of the tumor mass. Thrombin likely potentiates this process by virtue of its ability to induce proliferation of endothelial cells. In addition, thrombin has been shown to disrupt the normal intercellular endothelial cell contacts important in preventing cells and plasma factors from escaping or entering the microvasculature. The present hypothesis that thrombin may increase metastasis by disrupting these contacts is supported by studies demonstrating a correlation between decreased levels of anti-thrombin III (which removes thrombin and other proteases from plasma) and increased tumor metastasis.
Various studies have led the present inventors to conclude that high-affinity cell surface thrombin receptors (See Carney and Cunningham, Cell 15:1341, 1978) may be involved in tumor metastasis and angiogenesis. For example, studies have indicated that thrombin receptors can serve as binding sites for tissue plasminogen activator, a molecule secreted from metastatic tumor cells. Moreover, other studies demonstrate the involvement of tissue plasminogen activator in metastasis and angiogenesis. Thus, many of the effects of plasminogen activator may be mediated through its interaction with the cell surface thrombin receptor. It is therefore proposed that stimulation of the thrombin receptor serves to promote tumor metastases, while inhibition of the receptor will decrease metastatic activity.
Thrombin has also been shown to cause changes in the structure and function of cells which make up the endothelial vasculature. These studies suggest that thrombin may play a central role in the development of pulmonary edema as well as edema of other tissues. For example, thrombin has been shown to increase permeability of endothelial cell monolayers to macromolecules, to increase arterial pressure and pulmonary vascular resistance, to induce smooth muscle contraction, and to increase transcapillary fluid filtration. All of these effects may be mediated by thrombin""s interaction with cell surface thrombin receptors.
A number of recent studies have attempted to elucidate the mechanisms for thrombin-mediated cell stimulation. These studies have indicated to the present inventors that initiation of cell proliferation by thrombin requires two signals. The first signal appears to be dependent upon binding of the thrombin molecule to the high affinity cell surface thrombin receptor, while the second signal results from the enzymic activity of the thrombin molecule. Thus, unlike alpha-thrombin, neither DIP-alpha-thrombin (a proteolytically inactive thrombin derivative that retains receptor-binding activity) nor gamma-thrombin (an esterolytically active, but non-binding thrombin derivative) can initiate DNA synthesis or cell division. However, simultaneous addition of these two non-mitogenic thrombin derivatives initiates a level of DNA synthesis and cell division comparable to that initiated by alpha-thrombin.
These same thrombin derivatives have been used to distinguish intracellular events stimulated by high-affinity thrombin receptor occupancy from those resulting from proteolytic cleavage. Alpha-thrombin and gamma-thrombin both stimulate Na+/K+ ATPase activity, phospho-inositol turnover, and Ca2+ metabolism, whereas DIP-alpha-thrombin does not. Thus, these events are attributable to thrombin""s enzymic activity, not to receptor occupancy. Furthermore, these signals (the release of diacylglycerol and inositol triphosphate to cause Ca2+ mobilization) may in turn activate protein kinase C. Accordingly, it has been shown that phorbol myristate acetate (PMA), which activates protein kinase C, can substitute for enzymically active gamma-thrombin and initiate cell division in the presence of receptor saturating levels of DIP-alpha-thrombin or monoclonal antibody to the thrombin receptor. Thus, the requirements for enzymically active thrombin may indirectly relate to its activation of protein kinase C.
The precise signals generated by high-affinity interaction of thrombin with its receptor have been more difficult to define. However, it has recently been shown that DIP-alpha-thrombin stimulates a transient increase in intracellular cAMP. In contrast to ion fluxes and phosphoinositide turnover, cAMP levels are maximally stimulated by DIP-alpha-thrombin but are not stimulated by gamma-thrombin. Attempts to replace DIP-alpha-thrombin with cAMP analogs, however, have been unsuccessful. Therefore, it is possible that thrombin receptor occupancy produces a number of signals in addition to changes in cAMP levels.
One problem associated with the clinical application of thrombin directly to achieve such benefits is its susceptability to protease inhibitors by serum anti-thrombins. Such problems have heretofore prevented the use of thrombin in the clinic and has led the present inventors to identify smaller thrombin-active and thrombin antagonistic polypeptides which are not sensitive to the inhibitory effects of thrombin inhibitors.
The present invention provides for a number of smaller polypeptides which have been tailored to interact with the thrombin receptor to selectively stimulate or inhibit thrombin receptor occupancy related signals. It is believed that these polypeptides will prove to be useful in a wide variety of clinical settings where successful recovery may be influenced by thrombin receptor-mediated events.
The present invention provides a number of thrombin derivatives and methods useful for stimulating cell proliferation and promoting wound healing as well as methods useful in inhibiting wound healing, scar tissue formation, formation of tissue adhesions, and tumor metastasis and angiogenesis. The invention is based on the discovery that one may formulate polypeptide thrombin derivatives, or their physiologically functional equivalents, which selectively inhibit the interaction of thrombin with its high-affinity receptor or which mimic the stimulatory effects of thrombin.
Accordingly, the present invention, in its most general and overall scope, relates to synthetic or naturally derived polypeptide agonists and antagonists of thrombin receptor mediated events. Both of these classes of agents possess a thrombin receptor binding domain which includes a segment of the polypeptide that is capable of selectively binding to the high-affinity thrombin receptor. This segment of the polypeptide includes a sequence of amino acids homologous to a tripeptide cell binding domain of fibronectin.
In addition to the thrombin receptor binding domain, the stimulatory (agonistic) polypeptides possess a sequence of amino acids having sequences derived from the N-terminal amino acids of a dodecapeptide previously shown to be highly conserved among serine proteases. However, the inhibitory polypeptides do not include these serine esterase-conserved sequences.
The present invention is disclosed in terms of a showing that in the presence of a non-mitogenic (ie, non-stimulatory) concentration of alpha-thrombin, gamma-thrombin, or PMA, the interaction between stimulatory polypeptides and cell surface thrombin receptors provides the cell with a signal to proliferate. However, no proliferative signal results when cell surface thrombin receptors interact with the inhibitory polypeptides. Instead, the cells become more refractory to subsequent treatment with the stimulatory polypeptides. This result is believed to occur because the inhibitory polypeptides, which are themselves incapable of generating a proliferative signal, block binding of the stimulatory polypeptides.
As indicated above, practice of the cell-stimulatory methods of the present invention requires the presence of a secondary signal, for example, in the form of non-mitogenic concentrations of alpha-thrombin, gamma-thrombin, or PMA in order to supply the cells with the low-affinity proteolytic cleavage signal. Accordingly, the invention provides for pharmaceutical compositions and methods to which these compounds have been added. However, those of skill in the art will recognize that when the invention is practiced in vivo, native alpha-thrombin endogenous to the host will typically be adequate to provide this secondary signal.
Because thrombin is involved in a number of bioregulatory effects, the present invention, which allows one to selectively promote and inhibit these effects, has a number of clinical applications. For example, the invention provides a number of polypeptides useful in promoting wound healing. For such applications, the invention provides a polypeptide derivative of thrombin (or a functional equivalent of such a derivative) which has a thrombin receptor binding domain as well as a domain with a serine esterase conserved sequence of at least 12 amino acids. The invention also provides a polypeptide compound of at least 23 L-amino acids which has both a thrombin receptor binding domain and a domain with a serine esterase conserved amino acid sequence.
In one embodiment, the invention provides for several polypeptides containing specific amino acid sequences, such as a polypeptide compound in which the thrombin receptor binding domain includes the L-amino acid sequence Arg-Gly-Asp-Ala (SEQ ID NO: 1) together with the serine esterase conserved amino acid sequence, Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val (SEQ ID NO: 2). In a preferred embodiment, the polypeptide compound includes the L-amino acid sequence Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val (SEQ ID NO: 3).
The invention also provides for a pharmaceutical composition for promoting wound healing which includes of a therapeutically effective concentration of any of the compounds described above combined with a pharmaceutically acceptable excipient. Typically, such compositions include, for example, sufficient concentrations of the polypeptides to effect a stimulatory action on the thrombin receptor as demonstrated herein. Thus, such compositions should typically include sufficient concentrations to obtain levels of the polypeptides in the wound area which are shown in vitro to stimulate the receptor. When endogenous levels of a secondary signal are believed to be inadequate, compositions may be employed which further include the addition of a therapeutically effective concentration of alpha-thrombin or gamma-thrombin.
As used herein, a therapeutically effective concentration is defined as a concentration of the particular agent which provides a satisfactory increase in the rate of wound healing. Again, such concentrations are believed to correspond to levels sufficient to ellicit a stimulation of the thrombin receptor in vitro. However, it is believed that the compositions will prove most effective when the stimulatory (agonistic) polypeptides are-present at a concentration of from 0.1 uM to 10 uM.
Furthermore, where alpha-thrombin or gamma-thrombin are also employed, concentrations of from 0.1 uM to 10 uM are considered effective. However, empirical methods as are known in the art may be employed for determining more precisely the proper therapeutic dose for a given composition administered in a particular manner.
In addition, methods are provided which employ thrombin agonists to promote wound healing. One such method includes applying to the wound a therapeutically effective amount of a polypeptide derivative of thrombin, or a physiologically functional equivalent thereof, which has both a thrombin receptor-binding domain and a domain having a serine esterase conserved amino acid sequence. In general, thrombin is applied in amount sufficient to achieve fibroblast stimulation and thereby stimulate tissue regeneration. In that such methods typically involve topical application to a wound, possible sytstemic toxicity is not believed to be a problem. Therefore, virtually any concentration may be employed. However, in a preferred embodiment, the wound is treated to achieve a range of approximately 1 ng/cm2-10 ug/cm2 of wound surface.
The invention further provides a method for promoting wound healing in which a therapeutically effective amount of alpha-thrombin (1 ng/cm2-10 ug/cm2 of wound surface) or gamma-thrombin (1 ng/cm2-10 ug/cm2 of wound surface) is applied to the wound in conjunction with the foregoing thrombin derivatives. Of course, the specific polypeptides and pharmaceutical compositions provided by the invention may also be used in promoting wound healing. It is believed that these methods will be especially beneficial to patients involved in severe accidents (particularly burn patients), to those subjected to surgical procedures and to those with poor wound healing responses, such as aged and diabetic individuals.
Additional methods are provided for using the thrombin receptor inhibitory polypeptides. For example, the invention provides methods whereby scar tissue formation can be inhibited by administering to the wound or scar tissue, a therapeutically effective amount of a polypeptide derivative of thrombin, or a physiologically functional equivalent thereof, which has a thrombin receptor binding domain but does not have a serine esterase conserved sequence. Typically, such concentrations are adequate when sufficient to inhibit thrombin receptor mediated events. In a preferred embodiment, amounts ranging from 1 ng/cm2-10 ug/cm2 of wound surface are considered appropriate.
In a preferred embodiment, the polypeptide derivative of thrombin has the L-amino acid sequence Arg-Gly-Asp-Ala (SEQ ID NO: 1).
In general, these methods may be used in any situation where scar formation is undesirable, such as on burn patients or those subjected to opthalmic surgery. Moreover, the methods may also be of use in preventing keloidal scar formation. It is anticipated that spraying the wound with an aerosol spray will be a particularly sterile and efficacious manner of administering the polypeptide compound to the wounds of burn patients.
The inhibitory polypeptides should also prove useful in inhibiting the formation of tissue adhesions, defined as abnormal unions between body organs by formation of fibrous tissue. It is known that fibroblast proliferation is required for formation of such adhesions. Since alpha-thrombin is known to induce fibroblast proliferation, it follows that inhibition of thrombin-mediated mitogenesis by the peptides of the present invention could reduce adhesion formation. It is believed that administration of such inhibitory polypeptides to the surface of the affected organs will prove to be especially useful following certain surgical procedures, such as thoracic surgery, where gut adhesions often lead to postoperative complications.
It is further proposed that the inhibitory peptides will prove useful in the treatment of mammals with tumors to thereby inhibit tumor metastasis or anglogenesis. This view is supported by studies demonstrating that alpha-thrombin is able to disrupt normal inter-endothelial cell contacts important in preventing metastasis, as well as studies demonstrating that alpha-thrombin can induce the proliferation of endothelial cells required for angiogenesis. Accordingly, the invention provides a method whereby mammals with such tumors receive a therapeutically effective amount of a polypeptide derivative of thrombin, or a functional equivalent thereof, which has a thrombin receptor binding domain but does not have a serine esterase conserved sequence. While exact doses would need to be determined by empiracal methods known those skilled in the art, it is estimated that an amount sufficient to achieve a concentration of from 0.1 uM to 10 uM at the site to be treated is needed. Use of a polypeptide wherein the thrombin binding domain has an L-amino acid sequence Arg-Gly-Asp-Ala (SEQ ID-NO: 1) is specifically provided. It is contemplated that the polypeptides will be most efficacious in this regard when administered intravenously. However, other methods of administration will also likely prove to be effective.
In a most general embodiment, the invention provides for the use of inhibitory polypeptides to inhibit cell proliferation. This method encompasses, but is not limited to, situations in which one desires to inhibit cell proliferation in vitro. Of course, the inhibitory polypeptide, having a thrombin binding domain with the specific sequence Arg-Gly-Asp-Ala (SEQ ID NO: 1), may also be used as a general inhibitor of cell proliferation.
In another general embodiment, the invention comprises methods wherein the stimulatory polypeptides are used to potentiate cell growth. A polypeptide including the psequence Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val (SEQ ID NO: 3) is specifically provided. This method encompasses, but is not limited to, situations wherein one wishes to potentiate cell growth in vitro. Such cell-stimulatory uses may be potentiated by further providing an effective amount of alpha-thrombin (0.1 ug/ml-10 ug/ml), gamma-thrombin (0.1 ug/ml-10 ug/ml) or phorbol myristate acetate (10 ng/ml-100 ng/ml) in conjunction with the stimulatory polypeptide.
For purposes of the present invention, a thrombin derivative is defined as any molecule with an amino acid sequence derived at least in part from that of thrombin, whether synthesized in vivo or in vitro. Accordingly, a thrombin derivative, as referred to herein, designates a polypeptide molecule which comprises fewer amino acids than thrombin.
A physiologically functional equivalent of a thrombin derivative encompasses molecules which differ from thrombin derivatives in particulars which do not affect the function of the thrombin receptor binding domain or the serine esterase conserved amino acid sequence. Such particulars may include, but are not limited to, conservative amino acid substitutions and modifications, for example, amidation of the carboxyl terminus, acetylation of the amino terminus, conjugation of the polypeptide to a physiologically inert carrier molecule, or sequence alterations in accordance with the serine esterase conserved sequences.
A thrombin receptor binding domain is defined as a polypeptide sequence which directly binds to the thrombin receptor and/or competitively inhibits binding between high-affinity thrombin receptors and alpha-thrombin.
A domain having a serine esterase conserved sequence comprises a polypeptide A sequence containing at least 4-12 of the N-terminal amino acids of the dodecapeptide previously shown to be highly conserved among serine proteases (Asp-X1-Cys-X2-Gly-Asp-Ser-Gly-Gly-Pro-X3-Val) (SEQ ID NO: 8); wherein X1 is either Ala or Ser; X2 is either Glu or Gln; and X3 is either Phe, Met, Leu, His, or Val).
A stimulatory polypeptide is defined as a polypeptide derivative of thrombin, or a physiologically functional equivalent thereof, having the ability to both bind to and stimulate the thrombin receptor. Therefore, the stimulatory peptides will include both a thrombin receptor binding domain and a domain with a serine esterase conserved amino acid sequence.
An inhibitory polypeptide is defined as a polypeptide derivative of thrombin, or a physiologically functional equivalent thereof, having a thrombin receptor binding domain but without a serine esterase conserved amino acid sequence.